J Neurooncol (2015) 122:11–20 DOI 10.1007/s11060-014-1686-9
LABORATORY INVESTIGATION
Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells Yusuke S. Hori • Ryusuke Hosoda • Yukinori Akiyama • Rio Sebori • Masahiro Wanibuchi • Takeshi Mikami • Toshiya Sugino • Kengo Suzuki • Mitsuhisa Maruyama • Miki Tsukamoto • Nobuhiro Mikuni • Yoshiyuki Horio Atsushi Kuno
•
Received: 4 June 2014 / Accepted: 14 December 2014 / Published online: 21 December 2014 Ó Springer Science+Business Media New York 2014
Abstract Mitochondrial autophagy eliminates damaged mitochondria and decreases reactive oxygen species (ROS). The autophagy inhibitor chloroquine (CQ) potentiates temozolomide (TMZ) cytotoxicity in glioma cells, but it is not known whether CQ does this by inhibiting mitochondrial autophagy. The effects of CQ and TMZ on MitoSOX Red fluorescence, a mitochondrial ROS indicator, and cell death were examined in rat C6 glioma cells. Mitochondrial autophagy was monitored by the colocalization of MitoTracker Red fluorescence and EGFP-LC3 dots. Mitochondrial content was measured by MitoTracker Green fluorescence and immunoblotting for a mitochondrial protein. Finally, CQ’s effects on tumor cells derived from a glioblastoma patient and human U87-MG glioblastoma cells were assessed. TMZ (100–1,000 lM) alone
did not affect mitochondrial ROS or cell death in C6 cells, but when administered with CQ (10 lM), it increased mitochondrial ROS and cell death. Antioxidants significantly suppressed the CQ-augmented cell death in TMZtreated cells, indicating that mitochondrial ROS were involved in this cell death. TMZ treatment reduced MitoTracker Green fluorescence and mitochondrial protein levels, and these effects were inhibited by CQ. TMZ also increased the colocalization of EGFP-LC3 dots with mitochondria, and CQ enhanced this effect. CQ potentiated TMZ-induced cytotoxicity in patient-derived glioblastoma cells as well as human U87-MG glioblastoma cells. These results suggest that CQ increases cellular ROS and augments TMZ cytotoxicity in glioma cells by inhibiting mitochondrial autophagy.
Yusuke S Hori and Ryusuke Hosoda have equally contributed to this work.
Keywords Chloroquine Mitochondrial autophagy Mitochondrial ROS Glioma Cell death
Electronic supplementary material The online version of this article (doi:10.1007/s11060-014-1686-9) contains supplementary material, which is available to authorized users. Y. S. Hori R. Hosoda R. Sebori M. Maruyama M. Tsukamoto Y. Horio A. Kuno (&) Department of Pharmacology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan e-mail:
[email protected] Present Address: Y. S. Hori Asahi General Hospital, Asahi I-1326, Chiba 289-2251, Japan Y. Akiyama M. Wanibuchi T. Mikami T. Sugino K. Suzuki N. Mikuni Department of Neurosurgery, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan
Introduction The anti-malarial agent chloroquine (CQ) is reported to have potential as an adjuvant for glioblastoma treatment [1, 2]. As CQ’s anti-tumor mechanism, autophagy inhibition has attracted attention. Cell survival under various stresses is supported by autophagy, a lysosome-mediated process by which cells degrade and recycle proteins and organelles [3]. In tumor cells, autophagy also contributes to cell survival under chemotherapy [4], and blocking the autophagic process increases the efficacy of a variety of anti-cancer agents [5–8]. Autophagy is started by sequestration of cytosolic proteins and organelles into autophagosomes. To form an autophagosome, autophagy inducers, such as starvation, lead to activate the ULK1/2-Atg13-FIP200
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complex, which stimulates the beclin-1-Vps34-Atg14L complex. The microtubule-associated protein light chain 3 (LC3) plays essential roles in autophagosome formation by binding to phosphatidylethanolamine to form an LC3phosphatidylethanolamine conjugate, which is recruited to the autophagosome membrane [9]. The autophagosomes eventually fuse with lysosomes to form autolysosomes, where degradation occurs. CQ elevates the lysosomal pH, which inhibits both the fusion of autophagosomes and lysosomes and lysosomal protein degradation. Dysfunctional or damaged mitochondria, one of the main sources of intracellular reactive oxygen species (ROS) [10], are also removed by autophagic processes, which appear to be important for maintaining mitochondrial quality, and in turn, cellular function [11, 12]. Indeed, depleting autophagic proteins or treating cells with the autophagy inhibitor 3-methyladenine (3-MA) increases the mitochondrial ROS levels [13, 14]. However, it has not been elucidated whether inhibition of mitochondrial autophagy is involved in the CQ-mediated enhancement of cytotoxicity in glioma cells. In this study, we examined the effect of CQ on mitochondrial ROS levels and temozolomide (TMZ)-induced cell death in rat C6 glioma cells. We then analyzed whether mitochondrial autophagy occurs in TMZ-treated C6 cells to attenuate excess mitochondrial ROS, and whether CQ blocks this effect. Finally, we confirmed the effect of CQ on both cultured human glioma cells derived from a patient with glioblastoma and human U87-MG glioblastoma cells.
Materials and methods A detailed description of the materials and methods used in this study is provided in the Supplementary Materials and Methods. Cell culture Rat C6 glioma cells and human U87-MG glioblastoma cells were cultured in Dulbecco’s modified Eagle’s medium (Wako Pure Chemicals) supplemented with 1 % Antibiotic–Antimycotic Mixed Stock Solution (Nacalai Tesque, Kyoto, Japan) and 10 % fetal bovine serum (MP Biomedicals, Solon, OH, USA). Neurosphere culture from human glioblastoma tissue and measurement of cellular ATP content A tissue sample was obtained from a glioblastoma that was surgically resected from a patient treated at Sapporo Medical University Hospital. This study conformed to the
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principles outlined in the Declaration of Helsinki and was approved by the ethics committee of Sapporo Medical University. Written informed consent was received from the patient. Immunohistochemistry did not show any O(6) methylguanine-DNA methyltransferase (MGMT) expression and isocitrate dehydrogenase 1 (IDH1) mutations in the tumor. Neurosphere forming cells were obtained and cellular ATP content was measured as described in Supplementary Materials and Methods. Analysis of cell death Cell viability and apoptosis were assessed in C6 cells by a MuseTM Cell Analyzer using a Cell Count and Viability Assay Kit (Merck Millipore, Billerica, MA, USA) and nuclear condensation, respectively. In U87-MG cells, dead cells were also monitored by measuring the release of intracellular lactate dehydrogenase (LDH) into the incubation medium. Analysis of mitochondrial ROS level and mitochondrial content Mitochondrial ROS levels were monitored by using MitoSOX Red (Life Technologies) according to the manufacturer’s protocol. Mitochondrial content in C6 cells was measured by MitoTracker Green (MTG) fluorescence. Analysis of mitochondrial autophagy C6 cells were transfected with EGFP-LC3, and then stained with MitoTracker Red (MTR, 200 nM) after 42 h. The cells, either untreated or pretreated with CQ for 1 h, were incubated with vehicle or TMZ for 6 h. After fixation, the colocalization of EGFP-LC3 with mitochondria was analyzed by confocal laser microscopy. After mitochondria accumulate MTR in a membrane potential-dependent manner, the MTR fluorescence is retained even if mitochondrial potential is lost during fixation. The number of EGFP-LC3 dots colocalizing with MTR was counted in at least 30 randomly selected cells in each group from three independent experiments. Statistical analysis Results (mean ± SEM) were analyzed by Student’s twotailed t test for comparisons between two groups, or by one-way ANOVA and a Student-Neuman-Keuls post hoc test for comparisons among groups. Differences with a probability value less than 0.05 were considered significant. All analyses were performed with SigmaStat (Systat, San Jose, CA, USA).
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Fig. 1 CQ promotes mitochondrial ROS and cell death in TMZtreated C6 glioma cells. a Representative images of MitoSOX fluorescence in C6 glioma cells treated with vehicle (Control) or with 100 lM, 400 lM, or 1,000 lM temozolomide (TMZ100, TMZ400, or TMZ1000, respectively), with or without chloroquine (CQ), for 24 h. Scale bar: 10 lm. b MitoSOX fluorescence intensity. Average intensities of 18 fields from 3 independent experiments were compared. c Viability profiles of C6 cells treated with vehicle or
TMZ for 24 h, with or without CQ pretreatment. d The average percentage of dead cells, defined as cells that lost membrane integrity, in cells treated as in (c) (n = 4). e Representative images of Hoechst33342 staining in C6 glioma cells treated as in (c). Scale bar: 200 lm. f Percentage of apoptotic cells, indicated by condensed nuclei. Data from three independent experiments were compared. *P \ 0.05
Results
TMZ. Cytotoxicity was not evident in C6 cells treated with up to 10 lM CQ (Figure S1). Therefore, we used 10 lM CQ, a clinically achievable concentration [15], in the following experiments. Treatment with up to 1,000 lM TMZ alone increased neither mitochondrial ROS levels measured by MitoSOX Red fluorescence intensity (Fig. 1a, b) nor cell death, which was measured by both a Cell
CQ increases TMZ cytotoxicity via mitochondrial ROS in C6 glioma cells We first investigated whether CQ enhances mitochondrial ROS and cell death in rat C6 glioma cells treated with
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J Neurooncol (2015) 122:11–20 b Fig. 2 Mitochondrial ROS mediate promotion of cell death by CQ in
TMZ-treated C6 cells. a and b Representative images and quantification of MitoSOX fluorescence in C6 cells. Cells were treated with vehicle (Control) or TMZ (1,000 lM) for 12 h with or without CQ pretreatment. The effects of EUK-134 treatment (50 lM) were also analyzed. Average intensities of 15 fields from 3 independent experiments were compared. Scale bar: 100 lm. c Viability profiles of C6 cells treated with vehicle (Control), TMZ (1000 lM, 24 h), CQ, CQ and TMZ, or CQ and TMZ with pretreatment with the radical scavenger EUK-134 (EUK) (50 lM). d Average percentage of dead cells, treated as in (C) (n = 5). e Representative images of nuclear Hoechst33342 staining in C6 glioma cells treated with vehicle (Control), TMZ (4,000 lM), CQ, CQ and TMZ, or CQ with TMZ and NAC (200 lM). Scale bar: 200 lm. f Percentage of cells showing nuclear condensation, which marks apoptotic cell death. Data from three independent experiments were compared. g Beclin-1 mRNA levels in C6 glioma cells transfected with either control siRNA or siRNA against rat beclin-1 (n = 3). (h and i) C6 glioma cells transfected with control or beclin-1 siRNA and treated with vehicle or TMZ (4,000 lM), with or without N-acetylcysteine (NAC, 200 lM). h Representative images of Hoechst33342 nuclear staining and i the percentage of apoptotic cells indicated by nuclear condensation. Data from three independent experiments were compared. Scale bar: 200 lm. *P \ 0.05
Analyzer (Figure S2A) and nuclear condensation (Figure S2B). Co-administering CQ with various concentrations of TMZ significantly increased the mitochondrial ROS levels (Fig. 1a, b), with corresponding increases in cell death (Fig. 1c–f). CQ also enhanced the cell death in human neuroblastoma SH-SY5Y cells induced by TMZ although CQ alone did not affect cell death (Figure S3). We next examined whether the CQ-induced increase in mitochondrial ROS was involved in cell death. EUK-134, a synthetic superoxide dismutase/catalase mimetic, significantly suppressed the CQ-induced increase in MitoSOX Red fluorescence intensity in C6 cells treated with TMZ (Fig. 2a, b). Measurement of dead cells by Cell Analyzer showed that EUK-134 significantly suppressed cell death potentiated by CQ in C6 cells treated with TMZ (Fig. 2c, d). When cells were treated with 4,000 lM TMZ, prominent cell death was observed (Fig. 2e, f). CQ treatment further enhanced the cell death induced by 4,000 lM TMZ, and this enhancement of cell death was significantly suppressed by N-acetylcysteine (NAC), another antioxidant. These findings indicate that CQ enhanced the cell death in TMZ-treated C6 cells by upregulating mitochondrial ROS. We next investigated whether the knockdown of beclin1, a key molecule for autophagosome formation to initiate autophagy [9], mimics the CQ-induced augmentation of cell death, since mitochondrial ROS increase in cells with beclin-1 knockout or knockdown [13, 14]. While beclin-1 knockdown alone did not change the percentage of dead C6 glioma cells, it enhanced the TMZ-induced cell death (Fig. 2g–i); this enhancement was significantly reduced by NAC (Fig. 2h, i). These findings indicate that beclin-1 knockdown mimicked ROS-mediated enhancement of cell death induced by CQ.
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CQ blocks TMZ-induced mitochondrial autophagy We next examined whether TMZ and CQ induces mitochondrial autophagy in C6 cells. We first confirmed that treatment of C6 cells with TMZ increased autophagic flux as evidenced by p62 degradation (1,000 and 4,000 lM TMZ) and LC3-I to LC3-II conversion (400 lM*) by immunoblot analysis (Figure S4A). In the presence of a maximally effective concentration of CQ (50 lM) to inhibit autophagy, but not an incomplete dose of CQ (10 lM) [16], TMZ-induced p62 degradation was blocked and LC3-II accumulation was evident (Figure S4B). To monitor mitochondrial autophagy, we then analyzed the colocalization of autophagosomes with mitochondria, mitochondrial mass, and the effect of inhibiting autophagy flux by CQ on these parameters [17–19]. To visualize autophagosomes, we expressed EGFP-LC3 in C6 cells, and cells were also stained with MTR to label mitochondria. Colocalization of EGFP-LC3 dots with ‘‘fragmented’’ or ‘‘punctuate’’ MTR fluorescence but not overlays of EGFP-LC3 puncta over a larger region of MTR fluorescence was counted as autophagosome containing mitochondria [18]. Treatment with either TMZ or CQ significantly increased the colocalization of EGFPLC3 dots with fragmented mitochondria, and combining TMZ and CQ enhanced this effect (Fig. 3a, b). In addition, while amount of mitochondria indicated by MTG fluorescence was significantly decreased by TMZ (Fig. 3c), this reduction was suppressed by co-treatment with CQ (Fig. 3e, f). Immunoblot experiments also revealed that TMZ significantly decreased the level of cyclophilin D, a mitochondrial matrix protein, and CQ blocked this down-regulation (Fig. 3g, h). These findings strongly indicate that TMZ induced mitochondrial autophagy, and that CQ co-treatment blocked the elimination of mitochondria within the autophagosomes. To determine whether C6 cells induce mitochondrial autophagy in response to mitochondrial depolarization and ROS generation, we examined the effect of antimycin A (AntA), an inhibitor of mitochondrial complex III. AntA induces mitochondrial depolarization and increases mitochondrial ROS levels. AntA increased the number of autophagosomes and CQ enhanced this effect (Figure S5A, B). Similar to TMZ, AntA decreased mitochondria monitored by MTG fluorescence, and co-administration of CQ suppressed the reduction (Figure S5C, D). Treatment with AntA increased co-localization of EGFP-LC3 dots with MTR fluorescence, and co-treatment with CQ further increased the number of autophagosomes containing mitochondria (Figure S5E, F). These results indicate that C6 cells were capable of induction of mitochondrial autophagy in response to AntA treatment and that CQ blocked AntA-induced mitochondrial autophagy.
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Parkin protein was not detected in C6 glioma cells Parkin is reported to be recruited to damaged mitochondria to initiate mitochondrial autophagy [20]; however, parkin protein was not detected in C6 cells by immunoblots using either a mouse monoclonal anti-parkin antibody (Figure
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S6A) or a rabbit anti-parkin antibody (data not shown). Although parkin is reported to be upregulated in response to cellular stress [21], parkin protein was still undetectable under treatment with TMZ and CQ (Figure S6B). These data suggest that mitochondrial autophagy occurred independently of parkin in TMZ-treated C6 cells.
J Neurooncol (2015) 122:11–20 b Fig. 3 TMZ induces mitochondrial autophagy in C6 glioma cells.
a Representative and merged images showing EGFP-LC3 and MitoTracker Red (MTR) fluorescence in C6 cells. Cells were transfected with EGFP-LC3 and treated with vehicle (control), TMZ (4,000 lM), CQ, or CQ and TMZ for 6 h. Scale bar: 10 lm. Arrows indicate EGFP-LC3 dots colocalized with fragmented MTR signals. b Average number of EGFP-LC3 dots colocalized with MTR per C6 cell, treated as in (a) (n = 34–40 cells in each of three independent experiments.) c Representative images of MTG fluorescence in C6 cells treated with vehicle (control) or with 400 lM, 1,000 lM, or 4,000 lM TMZ (TMZ400, TMZ1000, or TMZ4000, respectively) for 6 h. Scale bar: 20 lm. d Average MTG fluorescence intensity per cell. Fifteen random fields from three independent experiments were recorded and analyzed. e Representative images of MTG fluorescence in C6 cells treated with vehicle (control), TMZ, CQ, or CQ and TMZ for 6 h. Scale bar: 20 lm. f Quantification of MTG fluorescence intensity per cell. Fifteen random fields from three independent experiments were recorded and analyzed. g A representative immunoblot for cyclophilin D (CypD) in C6 cells treated with vehicle or TMZ (4,000 lM) for 24 h, with or without CQ pretreatment. h Cyclophilin D protein levels, normalized to GAPDH (n = 3). *P \ 0.05
CQ enhances TMZ-induced cytotoxicity in human glioblastoma cells Finally, we examined whether CQ could enhance the TMZinduced mitochondrial ROS levels and cytotoxicity in human glioblastoma cells. Sphere-forming cells were isolated and cultured from a human MGMT-negative glioblastoma. These cells were positive for nestin, a neural progenitor-cell marker (Fig. 4a). CQ enhanced the TMZinduced increase in MitoSOX Red fluorescence in the glioblastoma-derived cells (Fig. 4b, c). Cell viability, evaluated by cellular ATP content, was significantly reduced by either 150 lM TMZ or 10 lM CQ, and was further reduced in TMZ-treated cells by CQ treatment (Fig. 4d). We also examined the effect of TMZ and CQ in human U87-MG glioblastoma cells. Although 300 lM TMZ alone did not induce cell death measured by LDH release, coadministration of 10 lM CQ with 100 or 300 lM TMZ significantly increased the percentage of dead cells (Fig. 5a). In the presence of another autophagy inhibitor bafilomycin A1 that blocks autophagy at the late stage by inhibiting lysosomal vacuolar type H?-ATPase, 300 lM TMZ was able to promote cell death in U87-MG cells (Fig. 5b), supporting the hypothesis that mitochondrial autophagy induced by TMZ plays a role in cytoprotection in glioblastoma cells. Although CQ or TMZ alone did not change MitoSOX fluorescence intensity, co-administration of CQ with TMZ significantly increased the intensity in U87-MG cells (Fig. 5c).
Discussion This study’s main finding was that a clinically achievable concentration of CQ enhanced TMZ cytotoxicity via
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increased mitochondrial ROS by suppressing the autophagy-mediated elimination of mitochondria in glioma cells. This is the first report to demonstrate that CQ enhances TMZ’s cytotoxicity by targeting mitochondrial autophagy. Furthermore, CQ also enhanced the cytotoxicity and mitochondrial ROS in human glioblastoma-derived tumor cells (Fig. 4) and human U87-MG glioblastoma cells (Fig. 5), demonstrating its clinical potential for treating glioblastoma. Lin et al. [22]. recently reported that treating U87 MG glioma cells with TMZ reduces mitochondrial mass, and that this reduction is partially reversed by 3-MA, which blocks autophagosome formation by inhibiting phosphatidylinositol-3 kinase. In that study, however, the mitochondrial mass was measured solely by 10-N-nonylacridine orange fluorescence, and it is not clear whether mitochondria were localized within autophagosomes [22]. In contrast, we showed that TMZ reduced the mitochondrial content and protein while increasing the localization of EGFP-LC3 to the mitochondria (Fig. 3). CQ, which inhibits lysosomes, blocked the TMZ-induced reductions in mitochondrial content and protein, and enhanced the TMZinduced colocalization of mitochondria with autophagosomes (Fig. 3). These multiple approaches clearly indicated that mitochondrial autophagy occurred in C6 cells in response to TMZ treatment. We also observed mitochondrial autophagy in C6 cells treated with AntA, which induces a collapse of mitochondrial membrane potential and increases mitochondrial ROS. These findings indicate that in response to genotoxic or mitochondrial stress, glioma cells can induces mitochondrial autophagy presumably to reduce cytotoxic mitochondrial ROS. Targeting of autophagy by CQ has attracted attention as adjuvant therapy for glioblastoma patients since Sotelo et al. reported the efficacy of adding CQ to conventional treatment in a randomized, double blind, and placebocontrolled trial [1]. Larger trials are warranted to confirm the benefit of CQ, and more than 20 clinical trials are ongoing to investigate the efficacy of CQ and the related molecule hydroxychloroquine combined with chemotherapies and radiation in various tumors (http://clinicaltrials. gov). To translate our findings into the clinical setting, we need to investigate how protective mitochondrial autophagy occurs in response to TMZ in glioma cells. Mitochondria appear to be degraded through non-selective macroautophagy and selective mitophagy [12]. Macroautophagy degrades a range of intracellular components, including mitochondria, while mitophagy specifically eliminates mitochondria. We showed that TMZ’s cytotoxicity was enhanced by siRNA-mediated beclin-1 knockdown (Fig. 2g–i), suggesting that beclin-1-mediated macroautophagy is involved in removal of mitochondria. It is reported that RNAi-mediated beclin-1 knockdown in
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Fig. 4 CQ enhances TMZ’s cytotoxic effect on neurosphere cells derived from human glioblastoma. a Representative images of nestin immunofluorescence in neurosphere cells derived from human glioblastoma tissue. b and c Representative images of MitoSOX Red fluorescence (b) and fluorescence intensity (c) in glioma cells treated with vehicle (Control), TMZ (150 lM), CQ (10 lM), or CQ and TMZ for 48 h. Data were average fluorescence intensity of 45 fields from three independent experiments. Scale bar: 100 lm. d Viability, measured by ATP content, of neurosphere cells treated with TMZ (150 lM) with or without CQ (10 lM). N = 6. *P \ 0.05
mouse embryonic fibroblasts increases the mitochondrial mass and blocks a hypoxia-induced reduction in mitochondrial mass [23]. In addition, inhibiting autophagy by knocking down either Atg5 or beclin-1 enhances cell death under hypoxia conditions via elevated intracellular ROS, suggesting that mitochondrial autophagy mediated by beclin-1 is an adaptive response to protect cells [23]. In addition, beclin-1 loss-of-function, whether by knockdown
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or knockout, increases mitochondrial ROS [13, 14]. These findings point to beclin-1 as a key molecule in the TMZinduced mitochondrial autophagy in C6 cells. There are limitations in this study. We cannot exclude the possibility that the effect of CQ was derived from blocking autophagy-mediated metabolic homeostasis. It has been reported that intracellular ATP level was increased in response to TMZ treatment via the autophagic
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Fig. 5 CQ and bafilomycin A1 enhance cytotoxicity of TMZ in human U87-MG glioblastoma cells. a Cell death measured by LDH release after 48 h of treatment of U87-MG cells with vehicle, 100 lM, or 300 lM temozolomide (TMZ), with or without 10 lM chloroquine (CQ) (n = 8). b Cell death measured by LDH release after 48 h of treatment of U87-MG cells with vehicle, 100 lM, or 300 lM TMZ, with or without 1 nM of bafilomycin A1 (Baf A1)
(n = 8). c Representative laser-confocal microscopic images of MitoSOX Red fluorescence (left) and average fluorescence intensity (right) in U87-MG cells treated with vehicle (Control), TMZ (300 lM), CQ (10 lM), or CQ and TMZ for 24 h. Scale bar: 25 lm. Data of fluorescence intensity per cell were analyzed from 14 filed in independent two experiments. *P \ 0.05. NS = not significant
process in glioblastoma cells [24]. Autophagy may also provide amino acids to support TCA cycle intermediates [25]. High levels of MGMT are significantly correlated with a poor prognosis in patients with glioblastoma [26]. Rat C6 glioma cells were resistant to TMZ alone at concentrations up to 1,000 lM (Fig. 1a) and were recently reported to express MGMT [27, 28]. Therefore, CQ may potentiate TMZ’s antitumor properties in MGMT-positive human glioblastomas. Further research is necessary to solve this issue. IDH mutations are generally thought to occur in lower grade glioma, and are associated with the favorable outcome and better response to temozolomide. Gilbert et al. recently reported p62 protein accumulation in both U87-MG cells overexpressing the R132H-mutated IDH1 and patient-derived IDH1-mutant tumors [29]. These findings suggest that the IDH1 mutation leads to inhibition of autophagic flux, resulting in the promotion of cell death. If so, better response to TMZ in IDH1 mutant tumors may be explained by attenuated autophagic activation. Cytotoxicity of CQ may be more potent in wild-type IDH1 glioblastomas with intact autophagy.
Conflict of interest of interest.
Acknowledgments This work was supported in part by grants for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 24791513) and from Adaptive and Seamless Technology Transfer Program through target-driven R&G, JST.
The authors declare that they have no conflict
Ethical standards All experiments were conducted in compliance with the ethics committee of Sapporo Medical University.
References 1. Sotelo J, Briceno E, Lopez-Gonzalez MA (2006) Adding chloroquine to conventional treatment for glioblastoma multiforme: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 144(5):337–343 2. Briceno E, Calderon A, Sotelo J (2007) Institutional experience with chloroquine as an adjuvant to the therapy for glioblastoma multiforme. Surg Neurol 67(4):388–391 3. Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451(7182):1069–1075 4. Amaravadi RK, Thompson CB (2007) The roles of therapyinduced autophagy and necrosis in cancer treatment. Clin Cancer Res 13(24):7271–7279 5. Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI et al (2007) Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 117(2):326–336 6. Han W, Sun J, Feng L, Wang K, Li D, Pan Q et al (2011) Autophagy inhibition enhances daunorubicin-induced apoptosis in K562 cells. PLoS ONE 6(12):e28491 7. Gini B, Zanca C, Guo D, Matsutani T, Masui K, Ikegami S et al (2013) The mTOR kinase inhibitors, CC214-1 and CC214-2,
123
20
8.
9. 10. 11. 12. 13.
14.
15.
16.
17.
18. 19.
J Neurooncol (2015) 122:11–20 preferentially block the growth of EGFRvIII-activated glioblastomas. Clin Cancer Res 19(20):5722–5732 Hu YL, DeLay M, Jahangiri A, Molinaro AM, Rose SD, Carbonell WS et al (2012) Hypoxia-induced autophagy promotes tumor cell survival and adaptation to antiangiogenic treatment in glioblastoma. Cancer Res 72(7):1773–1783 Kroemer G, Marino G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40(2):280–293 Balaban RS, Nemoto S, Finkel T (2005) Mitochondria, oxidants, and aging. Cell 120(4):483–495 Mizushima N, Levine B (2010) Autophagy in mammalian development and differentiation. Nat Cell Biol 12(9):823–830 Youle RJ, Narendra DP (2011) Mechanisms of mitophagy. Nat Rev Mol Cell Biol 12(1):9–14 Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC et al (2010) Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 12(3):222–230 Zhou R, Yazdi AS, Menu P, Tschopp J (2011) A role for mitochondria in NLRP3 inflammasome activation. Nature 469(7329):221–225 Ducharme J, Farinotti R (1996) Clinical pharmacokinetics and metabolism of chloroquine. Focus on recent advancements. Clin Pharmacokinet 31(4):257–274 Ni HM, Bockus A, Wozniak AL, Jones K, Weinman S, Yin XM et al (2011) Dissecting the dynamic turnover of GFP-LC3 in the autolysosome. Autophagy 7(2):188–204 Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, AcevedoArozena A, Adeli K et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8(4):445–544 Zhu J, Dagda RK, Chu CT (2011) Monitoring mitophagy in neuronal cell cultures. Methods Mol Biol 793:325–339 Ding WX, Yin XM (2012) Mitophagy: mechanisms, pathophysiological roles, and analysis. Biol Chem 393(7):547–564
123
20. Narendra D, Tanaka A, Suen DF, Youle RJ (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol 183(5):795–803 21. Zhang C, Lin M, Wu R, Wang X, Yang B, Levine AJ et al (2011) Parkin, a p53 target gene, mediates the role of p53 in glucose metabolism and the Warburg effect. Proc Natl Acad Sci U S A 108(39):16259–16264 22. Lin CJ, Lee CC, Shih YL, Lin CH, Wang SH, Chen TH et al (2012) Inhibition of mitochondria- and endoplasmic reticulum stress-mediated autophagy augments temozolomide-induced apoptosis in glioma cells. PLoS ONE 7(6):e38706 23. Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH, Wesley JB et al (2008) Mitochondrial autophagy is an HIF-1dependent adaptive metabolic response to hypoxia. J Biol Chem 283(16):10892–10903 24. Katayama M, Kawaguchi T, Berger MS, Pieper RO (2007) DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells. Cell Death Differ 14(3):548–558 25. White E (2012) Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer 12(6):401–410 26. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M et al (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352(10):997–1003 27. Ozawa A, Kadowaki E, Haga Y, Sekiguchi H, Hemmi N, Kaneko T et al (2013) Acetylcholine esterase is a regulator of GFAP expression and a target of dichlorvos in astrocytic differentiation of rat glioma C6 cells. Brain Res 1537:37–45 28. Kuo TC, Yang JS, Lin MW, Hsu SC, Lin JJ, Lin HJ et al (2009) Emodin has cytotoxic and protective effects in rat C6 glioma cells: roles of Mdr1a and nuclear factor kappaB in cell survival. J Pharmacol Exp Ther 330(3):736–744 29. Gilbert MR, Liu Y, Neltner J, Pu H, Morris A, Sunkara M et al (2014) Autophagy and oxidative stress in gliomas with IDH1 mutations. Acta Neuropathol 127(2):221–233
